HIV entry is mediated by the viral envelope glycoprotein, which comprises non-covalently associated surface (gp120) and transmembrane (gp41) subunits. gp120 is primarily involved in recognition of cellular receptors, while gp41 directly mediates membrane fusion. When peptides isolated from the gp41 N- and C-peptide regions (N- and C-peptides) are mixed in solution, they form a six-helix bundle, which represents the post-fusion gp41 structure (Lu 1995; Chan 1997; Weissenhorn 1997; Tan 1997). Three N-peptides form a central parallel trimeric coiled coil (N-trimer) surrounded by three antiparallel helical C-peptides that nestle into long grooves between neighboring N-peptides. The importance of this structure is indicated by the dominant negative inhibition of HIV entry by N- and C-peptides (Wild 1992; Jiang 1993; Eckert 2001).
The available inhibitory and structural data support a working model of HIV membrane fusion (FIG. 1) (Weissenhorn 1997; Eckert 2001; Chan 1998). Initially, gp120 interacts with cellular CD4 and a chemokine coreceptor (typically CXCR4 or CCR5), causing large conformational changes in gp120 that propagate to gp41 via the gp41-gp120 interface. gp41 then undergoes a massive structural rearrangement that unleashes its N-terminal fusion peptide, which embeds in the target cell membrane. At this stage of fusion, gp41 adopts an extended “prehairpin intermediate” conformation that bridges both viral and cellular membranes and exposes the N-trimer region. This intermediate is relatively long-lived (minutes) (Eckert 2001; Chan 1998; Furuta 1998), but ultimately collapses as the N- and C-peptide regions of each gp41 monomer associate to form a hairpin structure. Three such hairpins (trimer-of-hairpins) form the 6-helix bundle, which forces the viral and cellular membranes into tight apposition and leads to membrane fusion.
According to this model, an inhibitor that binds to the N-trimer and prevents hairpin formation can inhibit viral entry. This has been well supported by the discovery of numerous peptide, protein, and small molecule inhibitors that bind the N-trimer (Root 2004). A particularly interesting feature of the N-trimer is the deep hydrophobic “pocket” formed by its 17 C-terminal residues. This pocket has several enticing features as an inhibitory target including: (1) a very highly conserved sequence (Chan 1997; Eckert 1999; Root 2001), (2) an essential role in viral entry (Chan 1998), (3) a compact binding site vulnerable to inhibition by small molecules or short peptides, and (4) the availability of several designed peptides (e.g., IQN17 (Eckert 1999), IZN17 (Eckert 2001), 5-helix (Root 2001), NCCGN13 (Louis 2003) that authentically mimic the pocket structure.
Fuzeon is an approved HIV-1 entry inhibitor (also known as T-20 or enfuvirtide, Trimeris), which is a 36-residue C-peptide that binds to the N-trimer groove, but not the pocket (Wild 1994; Rimsky 1998). Although a significant breakthrough, Fuzeon has several serious limitations that have hampered its widespread clinical adoption, including high dosing requirements (90 mg, twice daily via injection), high cost (>$25,000 per patient per year), and the emergence of resistant strains both in vitro (Rimsky 1998) and in patients (Wei 2002). These problems have limited Fuzeon's clinical use to patients with multidrug resistant HIV-1 (salvage therapy).
Many of Fuzeon's limitations stem from protease sensitivity, a common problem for all L-peptide drugs. In contrast, D-peptide drugs have several theoretical advantages, including: (1) D-peptides are resistant to proteases (Milton 1992), a property that can dramatically increase serum half-life (Sadowski 2004), (2) L-peptides must be injected to avoid digestion, but short D-peptides can be absorbed systemically when taken orally (Pappenheimer 1994; Pappenheimer 1997), and (3) D-peptides represent a rich source of structural diversity because they can bind to targets with unique interface geometries not available to L-peptides. Despite these advantages, however, the potential of D-peptides has been largely unfulfilled.
Eckert et al. used mirror-image phage display (Schumacher 1996) to discover D-peptides that bind to the N-trimer pocket and inhibit HIV-1 entry with modest potency (Eckert 1999). These D-peptides provided the first direct proof that binding to the hydrophobic pocket is sufficient to block HIV-1 entry. Numerous other attempts to develop potent, pocket-specific entry inhibitors, include: minimized C-peptides (Judice 1997; Jin 2000; Sia 2002), helical mimics (Ernst 2002; Stephens 2005), and small molecules (Debnath 1999; Ferrer 1999; Zhao 2002; Jiang 2004; Frey 2006). However, at present, all of these inhibitors suffer from limited potency and/or toxicity in standard viral infectivity or cell-cell fusion assays.
What is needed in the art are peptides that can potently inhibit the entry of gp41 into cells.